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One object of the present invention is based upon the development and use
of a serum-free defined cell culture medium comprising a supplement
mixture, a component mixture, a vitamin mixture, an inorganic salt mixture
and amino acid mixture that avoids the problems inherent in the use of
serum. In particular, the defined medium is useful in culturing
fibroblasts, especially chondrocytes. Another object of the present
invention is to claim a method of enhancing the differentiation of
chondrocytes and enhancing the synthesis of a cartilage specific matrix
using tumor growth factor beta (TGF-.beta.). Another object of the present
invention is to claim a method of enhancing the differentiation of
chondrocytes using the combination of TGF-.beta.and IGF.

1. A method for enhancing the rate of re-differentiation of passaged, de-differentiated, human articular chondrocytes, comprising the step of culturing said passaged,
de-differentiated chondrocytes in a medium supplemented with TGF-.beta. and a growth factor selected from the group consisting of: IGF and insulin.

2. The method of claim 1, wherein the TGF-.beta. is TGF-.beta.1.

3. The method of claim 1, wherein the TGF-.beta. is TGF-.beta.2.

4. The method of claim 1, wherein the TGF-.beta. is present at 0.2 to 5.0 ng/ml.

5. The method of claim 1, wherein the growth factor is insulin.

6. The method of claim 1, wherein the growth factor is IGF.

7. The method of any one of claims 1-6, wherein the medium is defined.

10. The method of claim 5, wherein the medium is defined and comprises DME, human serum albumin, insulin and TGF-.beta..

11. A composition comprising passaged, de-differentiated human articular chondrocytes in a medium supplemented with TGF-.beta. and a growth factor selected from the group consisting of: IGF and insulin.

12. The composition of claim 11, wherein the TGF-.beta. is TGF-.beta.1.

13. The composition of claim 12, wherein the TGF-.beta. is TGF-.beta.2.

14. The composition of claim 12, wherein the TGF-.beta. is present at 0.2 to 5.0 ng/ml.

15. The composition of claim 11, wherein the growth factor is insulin.

16. The composition of claim 11, wherein the growth factor is IGF.

17. The composition of any one of claims 11-16, wherein the medium is defined.

18. The composition of claim 14, wherein the insulin is supplied by ITS.

20. The composition of claim 15, wherein the medium is defined and comprises DME, human serum albumin, insulin and TGF-.beta..

Description

BACKGROUND OF THE INVENTION

Initially, the successful culture of mammalian cells in vitro required supplementation of growth medium with serum which provides hormones and growth factors necessary for cell attachment and proliferation. Although serum is still widely used
for mammalian cell culture, there are several problems associated with its use (Freshney, Serum-free media. In Culture of Animal Cells, John Wiley & Sons, New York, 91-99, 1994): 1) serum contains many unidentified or non-quantified components and
therefore is not "defined"; 2) the composition of serum varies from lot to lot, making standardization difficult for experimentation or other uses of cell culture; 3) because many of these components affect cell attachment, proliferation, and
differentiation, controlling these parameters, or studying the specific requirements of cells with respect to these parameters, is precluded by the use of serum; 4) some components of serum are inhibitory to the proliferation of specific cell types and
to some degree may counteract its proliferative effect, resulting in sub-optimal growth; and 5) serum may contain viruses which may affect the outcome of experiments or provide a potential health hazard if the cultured cells are intended for implantation
in humans.

Primarily for research purposes, there has been some effort to develop biochemically defined media (DM). DM generally includes nutrients, growth factors, hormones, attachment factors, and lipids. The precise composition must be tailored for the
specific cell type for which the DM is designed. Successful growth in DM of some cell types, including fibroblasts, keratinocytes, and epithelial cells has been achieved (reviewed by Freshney,1994). However, attachment and proliferation of cells in DM
is often not optimal.

One potential application of defined medium is the expansion of chondrocytes released from adult human articular cartilage for treatment of cartilage defects with autologous chondrocyte transplantation (Brittberg et al, New England Journal of
Medicine, 331:889-895, 1994). Because this procedure involves the implantation of expanded chondrocytes into a patient, it may be desirable to avoid the use of serum or other undefined components during culture of the chondrocytes. For this
application, the DM would need to sustain proliferation of adult human articular chondrocytes seeded at low density until confluent cultures are attained.

Several investigators have reported proliferation of high density non-articular chondrocytes in DM (Kato et al, Exp. Cell Res., 125:167-174, 1980; Madsen et al, Nature, 304:545-547, 1983; Quarto et al, Bone, 17:588, 1995). Others have reported
proliferation of rabbit and human articular chondrocytes in DM (Boumedienne et al, Cell Prolif., 28:221-234, 1995; Schwartz, J. Cin. Chem. Clin. Biochem. 24:930-933, 1986). However, in these cases, chondrocytes were tested for growth in DM at high
density (.gtoreq.20,000 cells/cm.sup.2). Jennings and Ham (Cell Biology International Reports, 7:149-159, 1983) developed a serum-free medium for proliferation of chondrocytes isolated from costal cartilage of prepubertal humans and seeded at low
density. That medium required the use of polylysine-coated plates and included a liposome mixture for which the authors state that there are "inherent limitations in the degree of chemical definition".

Attempts to culture articular chondrocytes at sub-confluent densities in DM have not been successful. Adolphe et al (Exp. Cell Res., 155:527-536,1984) have developed a DM (Ham's F12 supplemented with insulin, transferrin, selenite, fibronectin,
bovine serum albumin, brain growth factor, fibroblast growth factor, hydrocortisone, and multiplication stimulating activity--now known as Insulin-like growth factor II) which supports proliferation of rabbit articular chondrocytes. However, they report
that serum-containing medium is necessary for the initial attachment of cells to the tissue culture vessel after seeding.

It has been reported that chondrocytes produce and secrete factors that promote their own attachment and proliferation (Shen et al, Endocrinology, 116:920-925, 1985). Examples include basic fibroblast growth factor (Hill et al, Growth Factors,
6:277-294, 1992), insulin-like growth factors (Froger-Gaillard et al, Endocrinology 124:2365-2372, 1989), transforming growth factor-.beta. (Villiger, P.M. et al., J. Immunol., 151:3337-3344, 1993), vitronectin, and possibly some unidentified factors
that promote their attachment and proliferation. Because articular cartilage is a non-vascularized tissue, and the chondrocytes embedded in cartilage have limited access to systemic growth factors, autocrine stimulation may play an important role in the
maintenance and proliferative capacity of these cells. To our knowledge, autocrine stimulation of chondrocytes has not been utilized for the purpose of enhancing the proliferation of human articular chondrocytes in DM.

During expansion in monolayer in vitro, articular chondrocytes de-differentiate, decreasing synthesis of matrix molecules normally produced by differentiated articular chondrocytes. It has been shown that for cells expanded in serum-containing
medium, this process can be reversed by transferring cells to a suspension culture system in the presence of serum (Benya and Shaffer, Cell, 30:215-224, 1982). If cells expanded in DM in monolayer are intended for implantation for healing of cartilage
defects (Brittberg et al, 1994), it is important to demonstrate they retain the potential to redifferentiate in suspension culture. A standard procedure for testing for redifferentiation potential is to suspend cells expanded in monolayer into agarose
and test for deposition of sulfated glycosaminoglycans by staining with safranin-.largecircle..

A need exists to standardize and control the proliferation and differentiation of adult human articular chondrocytes (HAC) cultured for any medical application, especially for application in humans.

SUMMARY OF THE INVENTION

One object of the present invention is based upon the development and use of a defined cell culture medium (serum-free) comprising a supplement mixture, a component mixture, a vitamin mixture, an inorganic salt mixture and amino acid mixture that
avoids the problems inherent in the use of serum. In particular, the defined medium is useful in culturing fibroblasts, especially chondrocytes.

Another object of the present invention is to claim a method for enhancing the differentiation of chondrocytes and for enhancing the synthesis of a cartilage specific matrix using tumor growth factor beta (TGF-.beta.).

Another object of the present invention is to claim a method for enhancing the differentiation of chondrocytes using a combination of TGF-.beta. and IGF.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention is based upon the development of a defined cell culture medium and culture method to standardize and control the proliferation and differentiation of human articular chondrocytes (HAC) cultured for implantation
into humans for repair of articular cartilage defects.

HAC were first cultured by plating the cells at 3000 cells per cm.sup.2 and allowing them to attach for one day in Dulbeccos Modified Eagles Medium (DMEM) supplemented 10% serum, then removing the serum-containing medium and refeeding with DMEM
basal medium in combination with a broad array of concentrations of the supplements described in Table 1. Every two to three days thereafter, cells were refed by completely replacing DM with fresh DM. Unable to induce cells to proliferate under these
conditions, different basal media were tried and found that a 1:1:1 ratio of DMEM:RPMI 1640:Ham's F12 (DRF) when combined with the supplements was effective in promoting cell proliferation after plating as above. HAC cultured in DRF+supplements
(complete DRF or cDRF) attained a terminal density equal or greater to the terminal density attained during culture in serum-supplemented medium. However, during the first several population doublings when the cell density was low, the proliferation
rate was slow relative to the rate in 10% serum. We demonstrated cell proliferation without providing the cells with serum for one day prior to addition of cDRF. However, the initial growth of these cultures was slow and variable. Further experiments
testing alternative supplements and different concentrations of the supplements failed to produce a cell culture system that consistently supported vigorous growth of HAC plated directly into DM without including serum at any step.

We had the idea that if we exchanged only half of the cDRF at refeeding, instead of completely exchanging the medium, factors secreted by the chondrocytes would boost their own proliferation. If the HAC secrete factor(s) required for growth in
cDRF, when the cell density is sparse, the quantity of the secreted factor may be close to the threshold requirement. This would explain the slow growth in early cultures and the variable results observed in the above experiments. Using the cDRF medium
combined with the approach of partial refeeding (described below), we obtained unexpectedly high yields of HAC in the complete absence of serum or any other undefined component after a brief time in culture. In the examples shown below, the volume of
cDRF used was reduced relative to the volume of serum-rich medium because presumably this would increase the concentration of secreted factors in the media and further promote proliferation. Later experiments indicate that reducing the volume is not
necessary and more optimal results may be achieved by using the larger volume while maintaining the practice of partial refeeding.

We have demonstrated that HAC cultured in suspension after expansion in cDRF generate colonies which stain positive with safranin-.largecircle.. This indicates that, during expansion in DM, the cells have not lost their capacity to produce
sulfated glycosaminoglycans, markers of chondrocyte differentiation. Because they retain their capacity to redifferentiate, chondrocytes expanded in cDRF may be suitable for autologous implantation for the purpose of healing cartilage defects.

Composition of cDRF

The culture medium, named cDRF, is composed entirely of commercially available and chemically defined basal media and growth supplements. cDRF is a modification of the DM developed by Adolphe et al (1984). As supplements to the basal media, we
have discovered that insulin transferin selenium (ITS) purchased from Collaborative Biomedical Products ((CBP) Bedford, Mass.), hydrocortisone purchased from Sigma (St. Louis, Mo.), basic fibroblast growth factor (FGF), fibronectin purchased from CBP
and insulin growth factor (IGF), both available from Genzyme Corporation (Cambridge, Mass.), are particularly useful in achieving the objectives of the medium described in this disclosure.

All materials are reconstituted, diluted, and stored as recommended by supplier. The three basal media, DMEM purchased from Gibco BRL, Grand Island, N.Y., Cat# 11965-084 (Table 2), RPMI DMEM purchased from Gibco BRL, Cat# 11875-051 (Table 2) and
Ham's F12 purchased from Gibco BRL, Cat#11765-021 (Table 2), are combined in a 1:1:1 ratio referred to hereinafter as DRF (Table 3). ITS, penicillin/streptomycin purchased from BioWhit-taker, and hydrocortisone are diluted into DRF and this medium is
stored up to 2 weeks at 2-8.degree. C. Basic FGF, IGF, and Fibronectin are diluted into the complete DRF medium (cDRF) on the day of use for cell culture.

Articular cartilage was harvested from femoral condyles of recently deceased human donors (age range: 29 to 53) within 24 hours of death and stored in isotonic media for up to 4 days at 2-8.degree. C. Chondrocytes (HAC) were released from the
cartilage by overnight digestion in 0.1% collagenase/DMEM. Remaining cartilage was further digested for 4 hours in 0.1% collagenase/0.25% trypsin/DMEM. The released cells were expanded as primaries in DMEM supplemented with 10% Fetal Bovine Serum, 100
U/ml Penicillin, and 100 ug/ml Streptomycin (serum-rich medium). At near confluence, cells were frozen in 10%DMSO/40% serum/50%DMEM.

For experiments performed with 2nd passage cells, ampules of frozen primaries were thawed, rinsed in media indicated below for initial seeding. For experiments performed in 3rd passage, cells were expanded through 2nd passage in serum-rich
media, harvested by trypsinization, and washed in seeding media as indicated.

The following disclosure describes the use of collagen matrices and the cytokine TGF-.beta. to enhance the redifferentiation and cartilage matrix formation process for dedifferentiated human articular chondrocytes. These findings are novel in
that the application demonstrates how the cytokine augments the re-expression of the differentiated chondrocyte phenotype for passaged and dedifferentiated human cells in a matrix rather than simply supporting the differentiated phenotype for
chondrocytes freshly released from cartilage tissue (primary cells) as others have shown (1,2). For those that have looked at the cytokine and its effect on re-expression, none have used it in a collagen matrix. The re-expression work centered on
either rabbit cells in an agarose matrix (3,4) or else the factors effect on human chondrocyte proliferation (5) and not differentiation specifically. This disclosure is a first demonstration that the use of the cytokine can augment the redifferentation
of the cells and enhance the rate at which new cartilage specific matrix is synthesized in the collagen sponge environment. This should enhance the ability of this system to regenerate new tissue, support increased mechanical loads and reform the
articular surface.

This disclosure describes a completely defined medium which will permit the re-expression of CII, a marker for chondrocyte differentiation, in a suspension of normal adult human articular chondrocytes that have de-differentiated as a consequence
of expansion in monolayer in vitro. It has been discovered that TGF-.beta.1 or .beta.2 and IGF-I satisfy the growth factor requirement for this differentiation process. This combination of growth factors in defined medium is potentially applicable to
improvements in the procedure of chondrocyte autologous transplantation (Bittberg et al, 1994). It may be used to prime chondrocytes for differentiation prior to implantation. Alternatively, it may be included as a supplement at the time of
implantation of the chondrocytes. This growth factor combination may also be used as a differentiation-stimulating supplement to chondrocytes embedded in a matrix intended for implantation into cartilage defects.

EXAMPLE 1

To test the concept of partial refeeding with cDRF, we compared chondrocyte growth by this new method with growth in culture conditions which we were familiar with: culture in Fetal Bovine Serum (FBS) or culture in cDRF after one day in FBS with
complete refeeding. Human chondrocytes from a 31 year old donor (HC31 cells) at 3rd passage were cultured under the conditions 1,2 & 3 described below.

Culture Condition 1 (FBS/complete refeeding)

Chondrocytes prepared as described above were seeded in triplicate into 10 cm.sup.2 tissue culture wells at a density of 3,000 cells per cm.sup.2 in 5 ml 10% FBS/DMEM and refed with 5 ml 10% FBS/DMEM one day after seeding and every 2-3 days
thereafter. At each refeeding, all media was removed and replaced with 5 ml of fresh medium.

Culture Condition 2 (FBScDRF/complete refeeding)

Chondrocytes were cultured as for condition 1 except that after one day in 10% FBS/DMEM, all refeedings were done with cDRF.

Culture Condition 3 (FBS-cDRF/partial refeeding)

Chondrocytes were cultured as for condition 1, except that after one day in 10% FBS/DMEM, all medium was removed and replaced with 3 ml cDRF. At each refeeding thereafter, partial refeeding was achieved by removing 1.5 ml of used cDRF and
replacing with 1.5 ml of fresh cDRF.

Cells were harvested at 7 and 13 days after seeding and samples were counted on a hemacytometer. The results in Table 4 show a marked enhancement of cell yield at 7 days and at 13 days in conditions of partial refeeding with cDRF compared to
that of complete refeeding.

We repeated example 1 but added in one more condition to determine whether we could eliminate the use of serum during the first day after seeding:

Culture Condition 4 (cDRF/partial refeeding)

Chondrocytes were cultured as for condition 1, except that they were seeded in 3 ml cDRF instead of 5 ml serum-rich media. At each refeeding, 1.5 ml of used cDRF was replaced with 1.5 ml of fresh cDRF.

Cells were harvested at 7 days after seeding. The results in Table 5 are generally similar to the results of example 1 for the three culture conditions that were repeated. Interestingly, the additional culture condition, in which direct plating
of cells into cDRF was combined with partial refeeding, yielded a yet higher quantity of cells.

Although there are only results for the 7 day timepoint in examples 2 & 3, the three examples combined are a strong indication that we can consistently attain cell densities of >100,000 cells/cm.sup.2 within two weeks of culture by thawing
frozen 1st passage cells, plating directly into defined medium without serum, and refeeding with half volumes.

EXAMPLE 4

We repeated example 2 again. In addition, we added one other condition to determine whether the partial refeeding method conferred an advantage to chondrocytes cultured in FBS:

Culture Condition 5 (FBS/partial refeeding)

Chondrocytes were cultured as for condition 1 except that at each refeeding, half the media was removed and replaced with fresh media.

The results in Table 7 are again consistent with previous examples showing a clear advantage of partial refeeding over complete refeeding when cDRF medium is used. In contrast, when serum-rich media is used, the partial refeeding method does not
increase and may decrease cell yields.

To assess the redifferentiation potential of chondrocytes after their expansion in monolayer culture in cDRF by the partial refeeding method, their capacity to form colonies in agarose which bind safranin-.largecircle. (Saf-.largecircle.
positive colonies) was assessed. Strain HC31 chondrocytes prepared as described above were thawed and seeded at 2nd passage into 225 cm.sup.2 tissue culture flasks (T225) at a density of 2,200 cells per cm.sup.2 in 100 ml cDRF per T225. Cells in cDRF
were refed by removing 50 ml (one-half the total volume) and replacing with 50 ml fresh cDRF. Refeeding was done one day after plating and every 2-3 days thereafter. As a positive control, parallel cultures were plated in 60 ml 10% FBS/DMEM per T225.
Cells in 10% FBS/DMEM were refed by removing the full volume of used medium and replacing with 60 ml fresh 10% FBS/DMEM. Four T225s were plated for each condition.

Cells were harvested by trypsinization from two T225's per culture condition at 12 and 14 days after seeding. At harvest, the cells were suspended at 2.5.times.10.sup.5 cells per ml in 10% FBS/DMEM and mixed 1:1 with 4% low-melt agarose. Four
ml of the cell/agarose suspension were plated onto a layer of 2 ml solidified high-melt agarose in 60 mm tissue culture dishes (P60). Platings were done in duplicate or triplicate. After solidification, the cultures were overlaid with 5 ml 10%
FBS/DMEM. The cultures were refed after 2-3 hours of equilibration and every 2-3 days thereafter until fixation.

After 3 weeks in agarose culture, the cells were fixed in 10% formalin and stained with safranin-.largecircle.. Saf-.largecircle. positive colonies of .gtoreq.2 .mu.m diameter were counted using a microscope. For each corresponding monolayer
condition, a total of 10 grids of 4 mm.sup.2 each were counted randomly from 2 P60s.

The results in Table 8 show that the capacity of HAC expanded in cDRF to generate Safranin-.largecircle. colonies after suspension in agarose is not statistically different than that of cells expanded in FBS. The similarity is clearer in the
results from the 14 day monolayer cultures which have a smaller sampling error. The reduction in the number of colonies generated after 14 days in monolayer, either FBS or cDRF, may be the consequence of maintaining the cells in monolayer in a
post-confluent state.

Primary chondrocytes were isolated from cartilage tissue from the femoral head of a 31 year old male. The cells were subcultured in monolayer. At third passage, the cells were seeded into a type-I collagen sponge matrix (Instat,
Johnson&Johnson) at 10.sup.7 cells/ml and cultured in DME media supplemented with either 10% fetal bovine serum (serum control), 1% ITS+media supplement (serum free control) or ITS+with TGF-.beta.1 at 1 or 5 ng/ml ("low dose" or "high dose",
Collaborative Biomedical Products, Bedford, Mass.). The DME media is standardly available, and may preferably include high glucose without sodium pyruvate. Differentiation state of the cells was determined by gene expression analysis with RNase
protection (Hybspeed RPA Kit, Austin Tex.) using 32P-labeled mRNA probes for type-I and type-II collagen and the cartilage specific proteoglycan Aggrecan. Matrix deposition was studied by use safranin-.largecircle./fast green stain as well monoclonal
antibody staining for collagen type-II and chondroitin sulfate. mRNA analysis of monolayer cells demonstrates type-I collagen expression with trace type-II and Aggrecan expression for the chondrocytes at the time of seeding. Upregulation of type-II
collagen with concurrent downregulation of type-I collagen expression was consistently observed for samples cultured in high dose TGF-.beta. supplemented and serum control cultures. In serum free and low dose TGF-.beta. conditions only modest type-I
collagen downregulation is observed. This concurrent expression behavior for type-I and type-II collagens is consistent with re-expression of the differentiated chondrocyte phenotype. By 4-weeks, an enhanced level of re-differentiation as shown by
RNase protection, was observed for samples cultured in high dose TGF-.beta. culture over the other groups. 8-weeks there was extensive proteoglycan staining throughout the thickness of the matrix for samples cultured in high dose TGF-.beta. conditions
demonstrated by both immuno- and histologic staining. For low dose TGF-.beta. and for serum and serum free controls, new matrix staining was relegated to the periphery or isolated pockets within the sponge matrix. This study demonstrates that passaged
human chondrocytes can re-express their differentiated phenotype in the type-I collagen sponge environment. The level of re-differentiation was demonstrated both at the genetic expression and at the matrix deposition level. TGF-.beta. modulated this
process by enhancing the rate of redifferentiation and the amount of new matrix deposition.

EXAMPLE 7

Confluent or near-confluent third passage adult human femoral condyle chondrocytes were harvested by trypsinization and suspended in alginate beads at a density of 10.sup.6 cells/ml. For each example described below, cells in alginate were
cultured at 37.degree. C., 9% CO.sub.2, in 25 mM HEPES buffered DMEM supplemented with 100U/ml penicillin, 100 .mu.g/ml streptomycin (basal medium), and additional supplements as indicated. Storage and dilution of supplements were performed as
recommended by suppliers. For each culture, 8 ml of alginate beads (8 million cells) were incubated in a 150 cm.sup.2 flask in 40 ml of the indicated media. Cultures were re-fed every 2-3 days. At timepoints indicated, cells were released from
alginate, pelleted, frozen and stored. RNA was isolated from the cell pellets and quantitated. The effect of the different culture conditions on the abundance of for collagen type I (CI) , collagen type II (CII), aggrecan (Agg) mRNAs was determined by
the Rnase protection assay, using 18 S rRNA (18 S) detection as an internal standard. The RNA probes used in the Rnase protection assay were transcribed from templates containing cDNA segments of the human genes for CI, CII, Agg, and 18 S. The culturing
of cells in alginate was done according to Guo, et al., Connective Tissue Research, 19: 277-297, 1989. The isolation of RNA was done according to manufacturer's instructions using the Qiashredder.TM. and RNeasy.TM. kits purchased from Qiagen
(Chatworth, Calif.). Rnase Protection assays were performed according to manufacturers instructions using Hybspeed.TM. RPA kit purchased from Ambion (Austin, Tex.).

Alginate culture and RNase protection assay protocols

OUTLINE

I. Expansion of chondrocytes in monolayer (1-2 weeks)

II. Culture of chondrocytes in alginate (1 week-6months)

A. Inoculation of cells into alginate beads

B. Refeeding

C. Harvesting cells from alginate beads

STOP POINT

III. Isolation, quantitation and aliquoting of cellular RNA (1-2 days; 1 day per set of RNA preps)

B. In vitro transcription from cDNA to prepare antisense radioactive RNA probes from cDNAs, followed by Dnase treatment to remove cDNA template (should be done within 3 days of Hybrization step, preferably the day before)

C. Gel purification of radioactive probes

D. Co-precipitation of cellular RNA with antisense probes (should be done the day before Hybrization step)

For R& D studies of chondrocyte differentiation, we cultured chondrocytes in alginate beads followed by detection of chondrospecific gene expression using RNase protection assays. The procedures, written in detail below, are derived from the
following sources;

Position a glass beaker containing 100 ml Isotonic Salt Solution under a Polytron mixer. Insert the end of the Polytron in the solution and run at high speed while very slowly adding 1.2 grams of alginate (Improved Kelmar, from Kelco) and moving
the beaker. After the alginate appears to be complete dissolved (.about.10-15 minutes), add magnetic stirbar and stir for .about.30 minutes. Autoclave 30 minutes, then run through 0.45 micron filter then 0.22 micron filter and store for up to six
months in the refrigerator.

Note: the basal medium affects the stability of the alginate beads; if using something other than DME as basal media, preliminary tests need to be done to test bead stability. See Guo et al (1989) Connective Tissue Res. 9:277

22 gauge needles and 10 ml syringes, one per alginate culture

25 mM HEPES -buffered DMEM, 120 ml per 8 ml alginate culture

T75 tissue culture flasks, one per alginate culture

Plastic bottle for suspension of cells in NaCl, one for each set of alginate cultures, maximum of 6 alginate cultures per set (capacity of .about.50-60 ml per monolayer T150 culture).

125 ml bottles, 1 per alginate culture

70 micron filters, 1 per alginate culture

Procedures

To avoid prolonged exposure of cells to 0.1 M Ca Cl.sub.2, prepare only .about.6 alginate cultures (.about.48 million cells) at one time from monolayer culture

13) Resuspend each tube of beads into 20 ml of respective media and transfer to labelled T162, then rinse remaining beads into flask with another 20 ml. (.about.8 million cells per T162, or somewhat less as .about.1 ml is lost during transfer
into syringe)

14) place loosely capped flasks in incubator.

B. Refeeding, every 2-3 days

For each alginate culture:

1) Stand flask on end, and tilt to allow beads to settle in one corner of flask.

Mix by pipetting and transfer to labelled 1.5 ml microfuge tubes Vortex each tube 30 seconds at high speed

Spin in microfuge momentarily

Mix briefly with pipet and transfer to QIA shredder

Spin at full speed in microfuge for 1 minute. If insoluble materials is visible in lysate (flowthru), microfuge lysate for 3 minutes at full speed and use only the supernatent for remaining steps. This second microfugation has not bee n
necessary to date.

5) RNeasy step 3:

Add 1 volume (600 .mu.l) 70% ethanol/DEPC-dH.sub.2 0 per sample and mix by pipetting. This lysate must not be centrifuged.

Add 700 .mu.l Wash Buffer RW1 into spin column, centrifuge as above and discard flow-thru. According to the trouble-shooting guide (p 23 of Rneasy manual), allowing the column to sit for 5 minutes after addition of RW1 and before centrifuging
may reduce DNA contamination

RNA is still in column. Add 500 .mu.l of Wash buffer RPE/Ethanol (1:4) to spin column.

Microfuge as above

Discard flow through

Add 500 .mu.l Wash buffer RPE/Ethanol (1:4) again to same spin column

Microfuge 2 minutes at full speed

Discard flow thru and collect on tube. Inspect for ethanol on outside of spin column and remove with kimwipe if necessary. Residual ethanol may interfere with subsequent steps. Transfer the spin column used above to a 1.5 ml collection tube
supplied with kit.

9) Elution of RNA from spin column: (RNeasy step 8)

Carefully add 30 .mu.l DEPC-treated water/0.1 mM EDTA per sample directly to membrane of the spin column, without touching the membrane with the pipet tip but making sure that the entire membrane is wetted

This reading would correspond to a total yield of 40 .mu.pg for a 50 .mu.l sample.

My yields have typically ranged from .about.8-80 .mu.g from 8 ml alginate cultures, depending on the conditions of culture. However, since I have done these preps, the methods for harvesting cells from alginate have been modified with the
intention of improving cell yield (specifically, the g-force during centrifugation of cells after dissolving alginate beads has been increased .about.5-fold).

i) 1.0 .mu.g RNA aliquots in DEPC-dH20/0.1 mM EDTA: For visualization of RNA on agarose gels or for Rnase Protection Assays, 1.0 .mu.g or less RNA is adequate. Because of the volume of the aliquots may be small (as little as 2 .mu.l) and because
of possible dessication of samples in freezer, do not subaliquot the 1.5 .mu.g aliquots stored in aqueous solution until they have been diluted into larger volumes after thawing. Use the 1.0 .mu.g aliquots once and discard any unused RNA after thawing.
According to the RNeasy Handbook, RNA stored in water at -80.degree. C., or even -20.degree. C., should be stable for at least a year.

ii) Remaining RNA stored in Ethanol: The samples stored in Ethanol should allow longer-term stability. This RNA must be treated as a suspension, not a solution. Before taking any sub-aliquots from these suspensions, they must be thoroughly
vortexed.

Ideally, it is better to precipitate the RNA, remove the ethanol, redissolve in aqueous solution, and requantitate before using.

5) RNA Ladder: Make one 2 .mu.l aliquot of RNA Ladder for each gel to be run and add 10 .mu.l 1.times.RNA Loading Buffer

6) Heat all of the RNA samples, including RNA Ladder at 65.degree. C. for 1 minute in heating block, vortex at setting 4-5 for 20 seconds, microfuge momentarily if sample splashes onto side of tube, heat at 65.degree. C. for 15 minutes, and
snap cool on ice.

Gel Electrophoresis

8) ufuge momentarily, and load 12 .mu.l of each sample per well, loading RNA Ladder into one well per gel. The quantity loaded will be 0.5 .mu.g per well except the RNA ladder which will be 2 .mu.g per well (.about.0.33 .mu.g/band). Freeze
remainder of samples in case samples need to be rerun.

Save photo for notebook. The 18S and 28S rRNAs should be clearly visible. Obvious downward smearing of the rRNA bands is indicative of RNA degradation. The intensity of the bands should be comparable among samples, if quantitation and
aliquoting were done properly.

Prancois Binette, using PCR technology, has synthesized all the human cDNA template used in these studies, with the exception of the 18S rRNA gene which is supplied by Ambion. These cDNAs are linked to .about.20 bp of the T7 phage promoter
oriented to promote synthesis of radioactive RNA from the human genes in the antisense orientation upon addition of T7 RNA polymerase and radioactive nucleoside triphosphates. Francois' maps of the human genes showing the probe positions and sizes are
attached.

The human cDNA templates currently available for these experiments include portions of the genes for:

Collagen Type I: chondrocyte dedifferentiation marker

Aggrecan, Collagen Types II & IX: chondrocyte differentiation markers

Collage Type X: chondrocyte hypertrophy marker

Day 1

B. In vitro transcription from cDNA to prepare antisense radioactive RNA probes from cDNAs, followed by Dnase treatment to remove cDNA template.

should be done within 3 days of Hybridization step, preferably the day before

The Rnase Protection Assay can be done using several probes combined, as long as the probes are different enough in size to allow separation during electrophoresis. However, each probe must be transcribed in separate tubes and gel purified from
different lanes of the gel before combining for the Rnase protection assay. In addition to preparing probes from the templates listed above, the 18S rRNA template should also be transcribed for every Rnase protection assay. Presumably, the quantity of
18S rRNA is equivalent among cells, independent of growth conditions, and is therefore a standard for comparing the amount of total cellular RNA used in the Rnase Protection Assays. Size marker RNA should also be used in each RNase Protection Assay.
Unlike the other probes, these can be transcribed as much as 2 months in advance of use and do not need to be gel-purified.

All components, except the templates and polymerase can be combined as one batch, then distributed as 13 .mu.l aliquots, one to each tube to receive template. Then add one template per tube, then polymerase

All procedures beginning with the addition of 32 P-CTP must be done using a plexiglass radiation shield

12) Wrap gel/backing with plasticwrap and expose x-ray film for 60 seconds, noticing orientation of gel and film (gel precisely in upperleft corner of film; dull side of film should be separated from gel by a single layer of plasticwrap)

13) Develop film, and using film as a guide, mark the location of the four bands containing the respective probes on the gel by adding mark directly to plasticwrap over gel with marking pen. Save film for X-ray film binder

14) With razor, cut out each band of the gel containing probe of interest, peal away plasticwrap, and drop each band into a separate 1.5 ml RNase-free tube of 300 .mu.l Elution buffer

15) Incubate gel in elution buffer for 2.5-3 hrs at 37.degree. C., then microfuge 1 minute at full speed

16) Add 10 ml scintillation fluid to each of four labeled scintillation vials

17) Add 3 .mu.l (1% of total volume) from each elution to separate scintillation vials

19) Calculate the volume in .mu.l, for each probe, equivalent to 25,000 counts

20) Combine probes into one 1.5 ml RNase-free tube, using above calculations to give total of 25,000 n CPM of each probe, where n equals the number of RNase protection assays (including controls) to be done with the probe mixture. Because the
energy from radiation can cause chemical breakdown of the RNA, the probes should be used within the next three days, the sooner the better

One 1.0 .mu.g aliquot of each cellular RNA sample to be probed (each in 650 .mu.l RNase-free tubes).

Ideally, this should include an aliquot or aliquots of cellular RNA which are known from previous assays to contain the RNA targeted by the probes being used (positive control) and one aliquot of cellular RNA from fibroblasts known not to express
chondrospecific genes (negative control).

Cold 100% Ethanol, 375 .mu.l per sample

Procedures

1) Combine (n+2)10 .mu.l Yeast RNA+(n+2)130 .mu.l Elution buffer in one tube where n=# of samples, including all controls, with volume of less than 10 .mu.l

2) To above mix, add (n+2)y .mu.l of probe mix from section C where n=# of samples, including all controls, with volume of less than 10 .mu.l where y .mu.l=volume of probe mix which contains 25,000 cpm of each probe

13) Digestion of non-hybridized ssRNA: At the end of the 10 minute hybridization, One tube at a time, transfer sample from 68.degree. C. bath directly to 37.degree. C. block and immediately add 100 .mu.l of diluted Rnase prewarmed to 37.degree. C.

Exception: To tube labeled "P+" (see Section D), do not add diluted Rnase. Instead, add 100 .mu.l of Digestion Buffer without Rnase. This sample will show the migration of intact probes during electrophoresis.

14) After all tubes have been treated as above, vortex each tube briefly and return to 37.degree. C. for 30 minutes, revortexing after the first 15 minutes. During this incubation, you may want to pour gel for electrophoresis (see below).

15) At end of 30' Rnase- treatment, add 150 .mu.l of cold Inactivation/Precipitation Mix to each tube.

Vortex and microfuge briefly. Transfer tubes to -20.degree. C. freezer for at least 15 minutes (Can leave for several hours).

1) Expose gel in erased phosphoimager cassette, recording position of gel on grid 2) After 1-4 days exposure, scan image and quantify bands

DNA fragments containing partial sequences of aggregan (Agg) (Doege et al., J. Biol. Chem 266:894-902, 1991) and types I and II collagens (Kuivaniemi et al., Biochem J. 252:633-640, 1988 and Baldwin et al., Biochem J. 262:521-528, 1989) were
generated by PCR amplification of human chondrocyte cDNA libraries. Paired oligonucleotides, representing coding sequences within each gene separated by several hundred basepairs (bp) were used as primers for PCR. Included at the 5' end of the
downstream primer, was an anchor sequence (CAGTGCCAT) for subsequent addition of the T7 RNA polymerase promoter. The sequence of the primers with upstream and downstream sequences shown respectively in 5' to 3' orientation are as follows:

The amplified fragments were inserted into pCRscript vector (Stratagene, Lajolla Calif.) for the propagation and maintenance. In order to generate templates for the transcription of antisense probes, a second PCR amplification was performed
using these cloned cDNA fragments. For priming, respective upstream primers shown above were each paired with the T7 promoter sequence containing the same anchor sequence (underlined) that use used in the first PCR amplification:

GGAATTCTTAGATAATACGACTCACTATAGGGCAGTGCCAT (SEQ ID No:9); DNA templates containing either 80 bp of the 18S rRNA gene or 316 bp of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, each linked to an upstream T7 promoter, were supplied by
Ambion (Austin, Tex.). Prior to use as a template, GAPDH sequence linked to the promoter was reduced to 149 bp by digestion with Dde I.

In vitro transcription from the above templates was performed using the Maxiscript.TM. kit (Ambion) according to manufacturer's instructions. Full-length probes were purified from the transcription reaction by electrophoresis on 7M urea, 4%
polyacrylamide 1.times.TBE gels, followed by autoradiography, excision from the gel of bands corresponding to the full length transcripts, and passive diffusion into probe elution buffer (supplied in the Maxiscript.TM. kit) for two hours at 37.degree.
C. The activity of the probe was quantified by scintillation counting.

RNase protection assays were performed using the Hybspeed.TM. RPA kit (Ambion) according to manufacturer's instructions. Briefly, radiolabelled antisense RNA probes for aggrecan and types I and II collagens were combined and hybridized with RNA
isolated from chondrocytes, using an excess of probe. A probe for 18S rRNA or GAPDH was also included in each hybridization mixture to normalize for total RNA. For negative controls, yeast RNA alone was combined with probes. For positive controls,
probes were hybridized to RNA samples know to contain sequences complementary to all four probes. Digestion with an RNaseA/RNase T1 mix was performed to degrade unhybridized RNAs. Hybridized RNAs protected from digestion were resolved by
electrophoresis as described above and visualized by autoradiography or by using a Fujifilm BAS-1500 phosphorimager. Bands on the phoshorimage representing types I and II collagen genes were quantified using MacBAS version 2.4 software. Any signal from
the corresponding position of the negative control (no chondrocyte RNA) was subtracted.

Cells in alginate culture were grown in the basal medium described above, supplemented as follows:

Culture 1: 1.times.ITS+

Culture 2: 1.times.ITS+and 0.2 ng/ml TGF-.beta.1

Culture 3: 1.times.ITS+and 1.0 ng/ml TGF-.beta.1

Culture 4: 1.times.ITS+and 5.0 ng/ml TGF-.beta.1

Cells were harvested at 7 and 21 days for RNA isolation.

The results of Rnase Protection Assay on RNA from the 7-day cultures showed that in the absence of TGF-.beta.1 (culture 1), there was little or no detectable CII or Agg mRNA while CI mRNA was abundant. With addition of 0.2 ng/ml TGF-.beta.1
(culture 2) there was a clear induction of mRNA abundance for the chondrocyte differentiation markers CII and Agg, while CI abundance was not significantly altered. Addition of higher TFG-.beta.1 concentrations (cultures 3 and 4) showed a dose-dependent
increase CII and Agg with no change in CI. Cultures harvested at 21 days yielded similar results. In a separate example we showed that a 100-fold molar excess of a monoclonal neutralizing antibody against TGF-.beta., when included with the culture
supplements listed for culture 3, yielded results similar to that of culture 1. This effectively eliminates the possibility that the differentiating activity was due to a contaminant of the TGF-.beta.1 preparation.

EXAMPLE 8

Culture conditions for cultures 1, 3 and 4 of example 7 were repeated. In parallel cultures, TGF-.beta.2 was used in place of TGF-.beta.1. The results from the TGF-.beta.1 and .beta.2 cultures were similar to the corresponding cultures from
Example 7, indicating that TGF-.beta.1 and .beta.2 have similar properties with respect to induction of chondrogenesis in this culture system.

EXAMPLE 9

Chondrocytes embedded in alginate were cultured in basal medium supplemented with ITS+and 1 ng/ml TGF-.beta.2 for 1,2,4,7, and 21 days. As a negative control, cells were cultured for 21 days in basal medium supplemented with ITS+alone. RNA
analysis of these cultures showed a general trend of increasing CII and Agg RNA throughout the first seven days (.about.5-fold increase in aggrecan and .about.40-fold increase in CII). At day 21, the abundance of CII and Agg mRNA apparently dropped off,
but remained high compared to day 1.

EXAMPLE 10

As shown in appendix C, 1.times.ITS+is a mixture of several components including 6.25 .mu.g/ml insulin. This example was performed to determine whether, in the above examples, the insulin in ITS+was playing a role in TGF-.beta. mediated
induction of CII and Agg. Secondly, if insulin was playing a role, we wanted to see if it can be replaced by IGF-I. The culture condition of culture 3 in example 7 was repeated. In parallel cultures, ITS+media was reproduced with insulin omitted or
replaced with 10 ng/ml IGF-I. The results from 7 day cultures showed that in the absence of insulin and IGF-I, 1 ng/ml TGF-.beta. induced neither CII nor Agg expression. However, addition to the culture of 10 ng/ml IGF-I in lieu of 6.25 .mu.g/ml
insulin, restored TGF-.beta.1 mediated induction of these chondrogenic markers to levels comparable to that of condition 3 of example 7. This suggests that the IGF receptor, which binds insulin with low affinity (Schmid, 1995), needs to be activated in
order for TGF-.beta. mediated chondrogenesis to occur. Furthermore, the use of IGF-I at approximately 600-fold lower concentration than that of insulin substantially reduces the possibility of contaminating factors affecting differentiation.

In a separate example, other components of the ITS+were omitted or substituted. We found that transferrin and selenious acid could be removed without consequence, and that human serum albumin can replace bovine serum albumin.

In conclusion, a complete defined medium, that includes basal medium supplemented with 1 ng/ml TGF-.beta.1 or .beta.2, 10 ng/ml IGF-I, 1 mg/ml human serum albumin, and may further include 5 .mu.g/ml linoleic acid, will induce de-differentiated
human chondrocytes to re-express the chondrocyte differentiated markers CII and Agg in suspension cultures.